1. Field of the Invention
The present invention relates generally to the fields of immunology and protein chemistry. More specifically, the present invention relates to a enhancement of tumor cell chemosensitivity and radiosensitivity using single chain secretory antibodies.
2. Description of the Related Art
Ovarian carcinoma is the leading cause of death from gynecologic cancer in the United States. Approximately 26,600 new cases were estimated to occur in 1995, resulting in 14,500 deaths from this disease. This figure exceeds the number of deaths from all other gynecologic malignancies combined. Over 70% of the patients present with late stage disease, the majority of which cannot be completely resected at the time of initial surgery. Chemotherapy has become the primary adjunct to surgery in obtaining a clinical remission or enhanced disease free survival in ovarian cancer patients. Although response to initial chemotherapy in ovarian cancer patients approaches 70%, most are transient and approximately 80% of patients (particularly those with advanced stage disease) will recur and eventually die of disease. Although a variety of salvage agents and strategies have been investigated, few have demonstrated long term effectiveness. In this regard, the five-year survival of patients with stage III disease remains, 15% to 30%.
Various approaches have been developed to accomplish gene therapy for cancer. There is increasing recognition that cancer results from a series of accumulated, acquired genetic lesions. To an ever larger extent, the genetic lesions associated with malignant transformation and progression are being identified. The recognition and definition of, the molecular basis of carcinogenesis makes it rational to consider genetic approaches to therapy. In this regard, a number of strategies have been developed to accomplish cancer gene therapy. These approaches include: 1) mutation compensation; 2) molecular chemotherapy; and 3) genetic immunopotentiation. For mutation compensation, gene therapy techniques are designed to rectify the molecular lesions in the cell having undergone malignant transformation. For molecular chemotherapy, methods have been developed to achieve selective delivery or expression of a toxin gene in cancer cells to achieve their eradication. Genetic immunopotentiation strategies attempt to achieve active immunization against tumor-associated antigens by gene transfer methodologies. Whereas the biology of each malignant disease target will likely dictate the approach taken, the majority of clinical gene therapy trials involve the genetic immunopotentiation approach. For most tumor types, however, the absence of clinical evidence of an anti-tumor effect has suggested the need for alternative approaches.
In addition to the gene therapy strategies discussed above, several reports have suggested that gene transfer approaches may be adjunctive to conventional chemotherapeutic modalities. In this regard, methods to enhance tumor cell conversion of cytotoxic prodrugs to their active forms have been developed. These include methods to enhance tumor cell metabolism of standard anti-tumor agents, such as oxazaphosphorines, by tumor cell transduction with cytochrome P-450. In another approach, transfer of viral or prokaryotic genes, such as the herpes simplex virus thymidine kinase (HSVTK), and E. coli cytosine deaminase are employed to sensitize tumor cells to the prodrugs ganciclovir or 5-fluorucytosine (5-FC), respectively, by conversion to toxic metabolites. In addition to these strategies, methods have been proposed based upon specifically reverting the molecular basis of the drug-resistant phenotype. This approach is based upon the concept that tumor cell drug resistance may be the result of diverse genetic alterations. These include mutational changes that lead to modifications in the structure of level of topoisomerase, to increased detoxification reactions, or to interference with the delivery of cytotoxic drug to intracellular targets. In addition, alterations affecting the regulation of the cell cycle and apoptosis are highly associated with drug resistance. These include inactivation of tumor suppressor genes, in particular p53 and Rb, and overexpression of proto-oncogenes such as those belonging to the myc family. Thus, based upon an understanding of the molecular basis of drug resistance, gene therapy strategies have been proposed to correct the genetic lesions etiologic of the drug resistant phenotype. To this end, augmentation of deficient tumor suppressor gene functions can restore tumor cell chemosensitivity. Roth et al. have shown that p53 gene replacement can enhance lung cancer chemosensitivity to cisplatin (CDDP). These studies establish the concept that gene transfer methods may be used in conjunction with conventional chemotherapeutic agents to achieve a synergistic antitumor effect. It is further suggested that specific rectification of the tumor cells genetic lesions can restore chemosensitivity.
Gene transfer approaches may be adjunctive to conventional radiation therapy. In this regard, methods to enhance tumor cell conversion of non-cytotoxic prodrugs to their active forms have been developed. The active forms of these drugs are potential or known radiosensitizers. One approach, transfer of viral or prokaryotic genes, such as herpes simplex thymidine kinase (HSVTK), and E. coli cytosine deaminase are employed to sensitize tumor cells to the prodrugs ganciclovir or 5-fluorocytosine (5-FC), respectively, by conversion to toxic metabolites. Both of these systems have also been employed to demonstrate enhanced radiation sensitivity. An alternative employed to enhance radiosensitivity in tumors involves the use of radiation inducible promoters to control gene expression. The tumor necrosis factor-xcex1 (TNFxcex1) gene under the control of the early growth response-1 (egr-1) promoter, was used to show radiosensitization in vitro and in vivo. In addition, to these strategies, methods have been proposed based upon specifically reverting the molecular basis of the radiation resistant phenotype. Alterations affecting the regulation of the cell cycle and apoptosis are highly associated with radiation resistance or sensitization. These alterations include inactivation of tumor suppressor genes, in particular p53 and Rb, and overexpression of proto-oncogenes such as those belonging to the ras and myc families, although this is not universal. Inactivating DNA DSB repair genes could be an effective method to dramatically increase the radiosensitivity of human tumor cell lines. Thus, based upon an understanding of the molecular basis of radiation sensitivity/resistance, gene therapy strategies may provide novel mechanisms to enhance radiation efficacy.
The erbB-2 oncogene is important to the malignant transformation of selected neoplasms including ovarian carcinomas. ErbB-2 is a 185 kDa transmembrane protein kinase receptor with homology to the family of epithelial growth factor receptors. Aberrant expression of the erbB-2 gene may play a role in neoplastic transformation and progression. Specifically, ectopic expression of erbB-2 is capable of transforming rodent fibroblasts in vitro. In addition, transgenic mice carrying either normal or mutant erbB-2 develop a variety of tumors, including neoplasms of mammary origin. Importantly, it has been shown that amplification and/or overexpression of the erbB-2 gene occurs in a variety of human epithelial carcinomas, including malignancies of the ovary, breast, gastrointestinal tract, salivary gland, and lung. In the context of ovarian carcinoma, a direct correlation has been noted between overexpression of erbB-2 and aggressive tumor growth with reduced overall patient survival. As erbB-2 overexpression may be a key event in malignant transformation and progression, strategies to ablate its expression would be therapeutic.
Overexpression of erbB-2 is associated with tumor cell chemoresistance. In addition to its direct role in neoplastic conversion, erbB-2 overexpression is associated with tumor cell resistance to chemotherapeutic agents. In this regard, heterologous overexpression of human erbB-2 accomplished by genetic transduction has been shown to increase the chemoresistance of murine fibroblasts and human lung carcinoma cells to a variety of chemotherapeutic agents. These findings are corroborated by the clinical observation that erbB-2 overexpressing tumors possess a higher intrinsic chemoresistance and thus are associated with a shorter relapse-free interval. Another line of evidence supporting the role of erbB-2 in modulating tumor cell chemoresistance is the observed therapeutic synergy between cisplatin and anti-erbB-2 monoclonal antibodies. These studies have documented that anti-erbB-2 antibodies capable of down-regulating the erbB-2 oncoprotein achieve enhanced tumor cell sensitivity to this chemotherapeutic agent. Thus, the erbB-2 oncoprotein plays a key role in determining tumor cell chemoresistance.
Therapeutic strategies for cancer have been developed which target the erbB-2 gene product. The association of overexpression of the erbB-2 gene product with neoplastic transformation and chemoresistance has led to the development of therapeutic strategies to down modulate erbB-2 levels in target tumor cells. Specifically, monoclonal antibodies (mAbs) have been developed which exhibit high affinity binding to the extracellular domains of the erbB-2 protein. A number of studies have demonstrated that a subset of these mAbs can elicit growth inhibition of erbB-2-overexpressing tumor cells, both in vitro and in vivo. In addition, a subset of these antibodies, which accomplish erbB-2 down-regulation enhance tumor cell chemosensitivity.
Gene therapy methods have been proposed to target erbB-2 overexpressing tumor cells to achieve down modulation of the oncoprotein. These approaches have included antisense strategies targeted to the transcriptional and post-transcriptional levels of gene expression. In the former instance, triplex-forming oligonucleotides binding the erbB-2 promoter region inhibit transcription of the erbB-2 gene. In addition, antisense oligonucleotides targeted to the erbB-2 transcript have accomplished phenotypic alterations in erbB-2 overexpressing tumor cells including down-regulation of cell surface expression and inhibition of proliferation.
Alternative methods to achieve targeted knockout of erbB-2 have been developed. In this regard, techniques have been developed to allow the derivation of recombinant molecules which possess antigen binding specificities expropriated from immunoglobulins. In this regard, single-chain immunoglobulin (sFv) molecules retain the antigen-binding specificity of the immunoglobulin from which they are derived, however, they lack other functional domains characterizing the parent molecule.
The Bcl-2-related protein family is an important regulator of programmed cell death or apoptosis. Members of this family with death antagonist properties include Bcl-2, Bcl-X, Bcl-w, Bfl-1, Brag-1, Mcl-1 and A1. Most of these proteins have to localize to the mitochondria to regulate apoptosis. Importantly, overexpression of death antagonist genes from the Bcl-2 family have been shown to protect a variety of cell lines from apoptosis induced by anti-cancer drugs. The Bcl-2 gene encodes a 26 kD protein that regulates apoptosis, at least in part, via its interaction with other members of the Bcl-2 family. Bcl-2 is mainly localized as an integral mitochondrial membrane protein, although Bcl-2 is also found to be associated with other membranes, including those of the endoplasmic reticulum (ER) and the nucleus. Extensive experimental evidence suggests that Bcl-2 promotes cell survival by preventing the onset of apoptosis induced by a wide variety of stimuli, including essentially all classes of anticancer drugs and x-irradiation. A role for Bcl-2 in cancer was initially identified in follicular lymphoma bearing the chromosomal translocation t(14;18) that juxtaposes the Bcl-2 gene with the immunoglobulin heavy chain locus, thereby up-regulating its expression.
Though first described in lymphoma, overexpression of Bcl-2 is also found in a number of non-hemopoietic cancers, including prostate cancer, breast cancer, and glioblastoma. In these cells, Bcl-2 may play an important role in protecting cancer cells from death induced by anti-cancer drugs. Estrogen-induced increases in Bcl-2 in the context of an estrogen-responsive human breast cancer cell line significantly enhanced their resistance to apoptosis, whereas antisense mediated reduction in Bcl-2 increased their sensitivity to anticancer drugs. Taxol-mediated inactivation of Bcl-2 by phosphorylation in prostate cancer cell lines renders them susceptible to apoptosis. Furthermore, Bcl-2 expression in ovarian cancer cells affects the cellular response to apoptosis and modulates their resistance to anti-cancer drugs. In addition to solid tumors, many non-Hodgkin lymphomas (NHL) and some acute myeloid leukemias (AMLs) often overexpress Bcl-2. Clinical studies of these hematological malignancies suggest an association between Bcl-2 expression, resistance to apoptosis, poor response to chemotherapy and shorter patient survival. Taken together, these results suggest a central role for Bcl-2 in the promotion of cell survival in solid and hematopoietic tumors.
Based upon these concepts, molecular therapeutic strategies to modulate Bcl-2 expression have been proposed. In this regard, antisense (AS) oligonucleotides targeted against Bcl-2 mRNA sequences and plasmid derived Bcl-2 AS transcripts have been shown to alter the growth and survival of lymphoid cells overexpressing Bcl-2 in vitro. In this context, several independent Bcl-2 AS studies have demonstrated a significant increase in apoptosis in treated cells, as well as more effective tumor cell killing following exposure to chemotherapeutic drugs. In vivo models have extended these findings, demonstrating that pre-treatment of lymphoma cells bearing the t(14;18) translocation with AS oligonucleotides to Bcl-2 mRNA inhibited the formation of tumors in a SCID mouse model. More recently, a clinical trial using Bcl-2 AS therapy in patients with NHL provided the first evidence of down-regulation of the Bcl-2 protein in humans.
BAG-1, a newly described Bcl-2 binding protein, functions in concert with Bcl-2 to prolong cell survival. In a human lymphoid cell line, gene transfer experiments have shown that coexpression of BAG-1 and Bcl-2 markedly enhanced protection from apoptosis induced by a variety of stimuli compared to cells transduced with either BAG-1 or Bcl-2 alone. In addition, overexpression of BAG-1 in liver progenitor cells increased hepatocyte growth factor (HGF)- and platelet-derived growth factor (PDGF)-induced protection from apoptosis. Thus, BAG-1 acts as a cell death inhibitor. Although the predicted amino-acid sequence of BAG-1 shares no homology with other proteins of the Bcl-2 family, it specifically interacts with Bcl-2 and can activate Raf-1 kinase. Of note, BAG-1 lacks the Bcl-2 family transmembrane domain and thereby localizes to the cytoplasm where it can interact with the cytoplasmic domain of the HGF and PDGF receptors. Despite these findings, the precise role of BAG-1 remains unclear, but the fact that it is expressed ubiquitously, and that it acts in conjunction with different growth factor receptors in preventing apoptosis, suggest that BAG-1 can function as a common adaptor protein between tyrosine kinase receptors and the anti-apoptotic machinery of the cell.
The prior art is deficient in the lack of effective means of enhancing tumor cell chemosensitivity to cancer drugs and enhancing sensitivity to radiation. The present invention fulfills this longstanding need and desire in the art.
In one embodiment of the present invention, there is provided a method of enhancing the chemosensitivity and radiosensitivity of a neoplastic cell, comprising the step of: introducing into the cell an antibody homologue, wherein the antibody homologue is secreted by the cell.
In another embodiment of the present invention, there is provided a method of enhancing the chemosensitivity and radiosensitivity of a neoplastic cell, comprising the step of: introducing into the cell an antibody homologue, wherein the antibody homologue is secreted by the cell; and contacting said cell with an anti-neoplastic agent, radiation or a combination thereof.
In another embodiment of the present invention, there is provided an antibody homologue, said antibody homologue is engineered so that said antibody homologue is secreted by a cell.
Other and further aspects, features, and advantages of the present invention will be apparent from the following description of the presently preferred embodiments of the invention given for the purpose of disclosure.